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Abstract [Objectives] This study was conducted to improve the efficiency of genetic transformation using growing points of corn sprouts.
[Methods] A mixed liquid of plant hormones 6BA and KT at a suitable concentration and ratio was added dropwise to the growing points of corn sprouts， followed by culture under appropriate conditions.
[Results] Corn sprouts could be induced to form multiple plants， thereby improving the transformation efficiency of growing points in corn sprouts.
[Conclusions] This study provides a breeding method using growing points of corn sprouts as transformation receptors for direct plantlet formation without tissue culture by the Agrobacterium transgenic technology.
Key words Corn; Sprout growing point; Differentiation; Plant
As an important food and feed crop[1]， corn can be improved for better yield or quality through genetic breeding. Transgenic technology allows us to effectively use foreign genes to improve varieties and create breakthrough germplasms[2]， and corn transgenic technology has become increasingly important for improving corn traits. Once the Agrobacterium transformation method becomes a conventional method for the development of transgenic corn， transgenic breeding will also become a conventional breeding approach[3]. The advantages of the Agrobacteriummediated seed germination method are no time limit for the explant sources， no need to establish a plant regeneration system and omission of complicated operations such as tissue culture[4]. The use of the growing points in corn sprouts as transformation receptors has been valued by people and has been used more and more widely. Li et al.[5]used corn shoot tips as the starting material to induce clumps of multiple shoots， followed by subculture， and with these clumps as receptors， a large number of transgenic plants were obtained by transforming herbicideresistant gene als into corn cells using a gene gun， indicating that the invitro shoot tip culture system is a good transgenic receptor system for corn[5]. However， these methods are still inseparable from the way of tissue culture， so they are subject to many restrictions. The transgenic method of transforming the growing points of corn sprouts directly into plantlets has many advantages. However， because transgenic plants obtained by using the growing points of corn sprouts as receptors are generally chimeras[6-8]， the current method of transforming the growing points of corn sprouts using Agrobacterium only can obtain one plant from the transformation of one growing point of corn sprout. Through the improvement of the culture technology， multiple plants were differentiated from a growing point of corn sprout. Due to the different locations of the differentiated sprouts， each plant obtained in this way generally originated from different cells， so that each plant could obtain different transformation events. In this way， transforming one corn sprout can obtain multiple different transgenic plants， which is of great significance for improving the transformation efficiency of growing points in corn sprouts. Materials and Methods
Experimental materials
Mature seeds of maize inbred line "Zheng 58" were used as an experimental material.
Experimental method
The method was basically carried out according to the method steps that we previously used to transform the growing points of corn sprouts with Agrobacterium into plantlets[6-8]. The specific steps were given as below.
Seed disinfection and germination
The corn seeds were cleaned and rinsed with sterile water for 5 min. The clean seeds were then disinfected with 0.1% HgCl2 for 12 min， followed by rinsing with sterile water 5 times for 2 min each time. The seeds were placed in petri dishes （specification： 9 cm in diameter） that had been sterilized at a high temperature with 2 layers of filter paper at the bottom according to 20 corn seeds per petri dish. Finally， 8 ml of sterile water was added into each dish， and the seeds were incubated in the dark at 23-25 ℃ for 3-4 d[6-8].
Collection of the growing points
A scalpel was used to remove coleoptile and young leaves from the corn shoots with a length of about 0.5 cm， to expose the growing points. The corn seeds were then placed in petri dishes that had been sterilized at a high temperature with 2 layers of filter paper at the bottom according to seven corn seeds per dish. Finally， 1 ml of sterile water was added into each dish， followed by covering the dish， and the seeds were incubated in the dark at 23-25 ℃ for 3-4 d[6-8].
Addition of plant hormones
To each growing point， 2.5 μl of a plant hormone mixture was dropped， with no plant hormones as a contrast. The concentrations of dropped 6BA and KT were 1 mg/ml， and the volumes of the plant hormones dropped to each treatment are shown in Table 1. Finally， 8 ml of sterile water was added into each dish， followed by covering the dish， and the seeds were incubated in the dark at 23-25 ℃ for 3-4 d.
Light culture
The petri dishes were uncovered， and sterile water was added to the petri dishes to 1/3 of the seeds. The seeds were then cultured in a condition with 12 h of illumination at 24-26 ℃ every day （Fig. 1）.
Plantlet training and transplanting
When the shoots grew to a length of 1-2 cm， and the corn plantlets were transplanted into vermiculite and cultivated for 3-4 d. After training the plantlets， they were transplanted into flower pots and cultivated under natural light at 25-26 ℃.
Results and Analysis According to the experimental results， treatment 3 was most beneficial to promote the growth of corn sprouts （Table 2）， that is， 0.5 μl of KT at a concentration of 1 mg/ml + 2μl of 6BA at a concentration of 1 mg/ml was beneficial to promote growth. After the above treatments， there were no multiple stems in the early stage after emergence， but after the plantlets were transplanted to flower pots， as the corn plantlets grew， the corn plantlets of treatment 3 had 2-4 shoots at the base of the stem （Fig. 2）， each of which could develop into a complete corn plant， such that more corn plants could be obtained from one corn seed. Due to the effects of plant hormones， some corn leaves were curled. This deformity phenomenon was more obvious in the early stage， but the leaves gradually became normal in the later stage of growth， and there was no effect on pollination and seed set of corn basically.
Conclusions and Discussion
Each corn sprout could be induced to form multiple plants through the addition of plant hormones 6BA and KT at appropriate concentrations and proportions to the growing points of corn sprouts， followed by culture. Therefore， a breeding method using the growing points of corn sprouts as transformation receptors by the Agrobacterium transgenic technology was provided， without the need for tissue culture. By the method， more transformed plants could be obtained through the transformation of the growing point of a corn sprout， thereby improving the transformation efficiency of the growing points in corn sprouts.
References
[1] LIU YJ， JIA ZW， LIU Y， et al. Establishment and application of largescale transformation systems for maize[J]. Scientia Agricultura Sinica， 2014， 47（21）： 4172-4182
[2] BAI YF， WANG GY， GOU MY， et al. Influencing factors on maize transgene process mediated by Agrobacterium[J]. Chinese Journal of EcoAgriculture， 2006， 14（3）： 173-175. （in Chinese）
[3] WANG HW， LI FH， WANG ZB， et al. Research and developments in transgenic maize[J]. Journal of Maize Sciences， 2006， 14（4）： 17-20. （in Chinese）
[4] ZHANG LJ， LIU LL， ZHANG JZ， et al. Trauma embryo of genetic transformation system with Agrobacteriummediated method[J]. Journal of Anhui Agricultural Sciences， 2015， 43（3）： 25- 28. （in Chinese）
[5] LI GS， ZHANG QW， ZHANG JR， et al. Establishment of clusteredsprout system of maize and regeneration of herbicideresistant transgenic plants[J]. Science China， 2001， 31（5）： 385-391. （in Chinese）
[6] LYU MYU， DONG FS， ZHANG JM， et al. Effect of low temperature treatment on Agrobacteriummediated transformation of maize seed buds[J]. Agricultural Biotechnology， 2018， 7（3）： 75-76.
[7] LYU MY， DONG FS， ZHANG JM， et al. Effects of ultrasonic treatment of maize seed sprouts on Agrobacterium transformation[J]. Journal of Maize Sciences， 2018， 26（5）： 54-57. （in Chinese）
[8] LYU MY， DONG FS， ZHANG JM， et al. Effect of cocultured water state on Agrobacterium transformation of maize[J]. Tianjin Agricultural Sciences， 2018， 24（12）： 8-10. （in Chinese）
[Methods] A mixed liquid of plant hormones 6BA and KT at a suitable concentration and ratio was added dropwise to the growing points of corn sprouts， followed by culture under appropriate conditions.
[Results] Corn sprouts could be induced to form multiple plants， thereby improving the transformation efficiency of growing points in corn sprouts.
[Conclusions] This study provides a breeding method using growing points of corn sprouts as transformation receptors for direct plantlet formation without tissue culture by the Agrobacterium transgenic technology.
Key words Corn; Sprout growing point; Differentiation; Plant
As an important food and feed crop[1]， corn can be improved for better yield or quality through genetic breeding. Transgenic technology allows us to effectively use foreign genes to improve varieties and create breakthrough germplasms[2]， and corn transgenic technology has become increasingly important for improving corn traits. Once the Agrobacterium transformation method becomes a conventional method for the development of transgenic corn， transgenic breeding will also become a conventional breeding approach[3]. The advantages of the Agrobacteriummediated seed germination method are no time limit for the explant sources， no need to establish a plant regeneration system and omission of complicated operations such as tissue culture[4]. The use of the growing points in corn sprouts as transformation receptors has been valued by people and has been used more and more widely. Li et al.[5]used corn shoot tips as the starting material to induce clumps of multiple shoots， followed by subculture， and with these clumps as receptors， a large number of transgenic plants were obtained by transforming herbicideresistant gene als into corn cells using a gene gun， indicating that the invitro shoot tip culture system is a good transgenic receptor system for corn[5]. However， these methods are still inseparable from the way of tissue culture， so they are subject to many restrictions. The transgenic method of transforming the growing points of corn sprouts directly into plantlets has many advantages. However， because transgenic plants obtained by using the growing points of corn sprouts as receptors are generally chimeras[6-8]， the current method of transforming the growing points of corn sprouts using Agrobacterium only can obtain one plant from the transformation of one growing point of corn sprout. Through the improvement of the culture technology， multiple plants were differentiated from a growing point of corn sprout. Due to the different locations of the differentiated sprouts， each plant obtained in this way generally originated from different cells， so that each plant could obtain different transformation events. In this way， transforming one corn sprout can obtain multiple different transgenic plants， which is of great significance for improving the transformation efficiency of growing points in corn sprouts. Materials and Methods
Experimental materials
Mature seeds of maize inbred line "Zheng 58" were used as an experimental material.
Experimental method
The method was basically carried out according to the method steps that we previously used to transform the growing points of corn sprouts with Agrobacterium into plantlets[6-8]. The specific steps were given as below.
Seed disinfection and germination
The corn seeds were cleaned and rinsed with sterile water for 5 min. The clean seeds were then disinfected with 0.1% HgCl2 for 12 min， followed by rinsing with sterile water 5 times for 2 min each time. The seeds were placed in petri dishes （specification： 9 cm in diameter） that had been sterilized at a high temperature with 2 layers of filter paper at the bottom according to 20 corn seeds per petri dish. Finally， 8 ml of sterile water was added into each dish， and the seeds were incubated in the dark at 23-25 ℃ for 3-4 d[6-8].
Collection of the growing points
A scalpel was used to remove coleoptile and young leaves from the corn shoots with a length of about 0.5 cm， to expose the growing points. The corn seeds were then placed in petri dishes that had been sterilized at a high temperature with 2 layers of filter paper at the bottom according to seven corn seeds per dish. Finally， 1 ml of sterile water was added into each dish， followed by covering the dish， and the seeds were incubated in the dark at 23-25 ℃ for 3-4 d[6-8].
Addition of plant hormones
To each growing point， 2.5 μl of a plant hormone mixture was dropped， with no plant hormones as a contrast. The concentrations of dropped 6BA and KT were 1 mg/ml， and the volumes of the plant hormones dropped to each treatment are shown in Table 1. Finally， 8 ml of sterile water was added into each dish， followed by covering the dish， and the seeds were incubated in the dark at 23-25 ℃ for 3-4 d.
Light culture
The petri dishes were uncovered， and sterile water was added to the petri dishes to 1/3 of the seeds. The seeds were then cultured in a condition with 12 h of illumination at 24-26 ℃ every day （Fig. 1）.
Plantlet training and transplanting
When the shoots grew to a length of 1-2 cm， and the corn plantlets were transplanted into vermiculite and cultivated for 3-4 d. After training the plantlets， they were transplanted into flower pots and cultivated under natural light at 25-26 ℃.
Results and Analysis According to the experimental results， treatment 3 was most beneficial to promote the growth of corn sprouts （Table 2）， that is， 0.5 μl of KT at a concentration of 1 mg/ml + 2μl of 6BA at a concentration of 1 mg/ml was beneficial to promote growth. After the above treatments， there were no multiple stems in the early stage after emergence， but after the plantlets were transplanted to flower pots， as the corn plantlets grew， the corn plantlets of treatment 3 had 2-4 shoots at the base of the stem （Fig. 2）， each of which could develop into a complete corn plant， such that more corn plants could be obtained from one corn seed. Due to the effects of plant hormones， some corn leaves were curled. This deformity phenomenon was more obvious in the early stage， but the leaves gradually became normal in the later stage of growth， and there was no effect on pollination and seed set of corn basically.
Conclusions and Discussion
Each corn sprout could be induced to form multiple plants through the addition of plant hormones 6BA and KT at appropriate concentrations and proportions to the growing points of corn sprouts， followed by culture. Therefore， a breeding method using the growing points of corn sprouts as transformation receptors by the Agrobacterium transgenic technology was provided， without the need for tissue culture. By the method， more transformed plants could be obtained through the transformation of the growing point of a corn sprout， thereby improving the transformation efficiency of the growing points in corn sprouts.
References
[1] LIU YJ， JIA ZW， LIU Y， et al. Establishment and application of largescale transformation systems for maize[J]. Scientia Agricultura Sinica， 2014， 47（21）： 4172-4182
[2] BAI YF， WANG GY， GOU MY， et al. Influencing factors on maize transgene process mediated by Agrobacterium[J]. Chinese Journal of EcoAgriculture， 2006， 14（3）： 173-175. （in Chinese）
[3] WANG HW， LI FH， WANG ZB， et al. Research and developments in transgenic maize[J]. Journal of Maize Sciences， 2006， 14（4）： 17-20. （in Chinese）
[4] ZHANG LJ， LIU LL， ZHANG JZ， et al. Trauma embryo of genetic transformation system with Agrobacteriummediated method[J]. Journal of Anhui Agricultural Sciences， 2015， 43（3）： 25- 28. （in Chinese）
[5] LI GS， ZHANG QW， ZHANG JR， et al. Establishment of clusteredsprout system of maize and regeneration of herbicideresistant transgenic plants[J]. Science China， 2001， 31（5）： 385-391. （in Chinese）
[6] LYU MYU， DONG FS， ZHANG JM， et al. Effect of low temperature treatment on Agrobacteriummediated transformation of maize seed buds[J]. Agricultural Biotechnology， 2018， 7（3）： 75-76.
[7] LYU MY， DONG FS， ZHANG JM， et al. Effects of ultrasonic treatment of maize seed sprouts on Agrobacterium transformation[J]. Journal of Maize Sciences， 2018， 26（5）： 54-57. （in Chinese）
[8] LYU MY， DONG FS， ZHANG JM， et al. Effect of cocultured water state on Agrobacterium transformation of maize[J]. Tianjin Agricultural Sciences， 2018， 24（12）： 8-10. （in Chinese）